Myelodysplastic Syndromes International Foundation Research Articles

Aberration Of SF3B1 Results In Deregulated Splicing Of Key Genes and Pathways In Myelodysplastic Syndromes

The recent discovery of a variety of somatic splicesomal mutations in the myelodysplastic syndromes (MDS) has revealed a new leukaemogenic pathway involving spliceosomal dysfunction. Pre-mRNA splicing proceeds by way of two phosphoester transfer reactions and is catalyzed by the spliceosome, which consists of the U1, U2, U4/U6, and U5 small nuclear ribonucleoproteins (snRNPs) and numerous non-snRNP proteins. The snRNPs are involved in recognising short conserved sequences of the pre-mRNA, including the 5′ and 3′ splice sites and the branch site, and in positioning the reactive nucleotides for catalysis. The spliceosome is a dynamic molecular machine, undergoing several major structural rearrangements during its functional cycle. Mutation of the Splicing Factor 3B, subunit 1 (SF3B1) gene is common in MDS, occurring in over 70% of patients whose disease is characterised by ring sideroblasts (RARS). The close association between SF3B1 mutation and ring sideroblasts is consistent with a causal relationship, and makes this the first gene to be strongly associated with a specific feature of MDS.Sf3b1 heterozygous knockout mice show the presence of ringed sideroblasts. In order to investigate the role of SF3B1 haploinsufficiency in MDS we have silenced SF3B1 using siRNA in the myeloid cell lines K562, TF-1, SKM1, HeL and OCIM2. Cell growth was impaired in all the cell lines with SF3B1 knockdown. Using Flow Cytometry, cell cycle analysis showed a significant increase in cells in the sub-G0 phase as well as G2/M arrest in the cell lines. We also observed impaired erythroid differentiation in hemin treated K562 and TF-1 cell lines with SF3B1 knockdown.Gene expression profiling (GEP) was performed in two cell lines with SF3B1 knockdown (K562 and TF1). Deregulated pathways and gene ontology categories included cell cycle regulation and alternative splicing using Ingenuity Pathway Analysis. We next performed Gene Set Enrichment Analysis (GSEA). The GSEA showed a significant enrichment of nonsense-mediated mRNA decay (NMD) genes that were up-regulated in cells with SF3B1 knockdown, suggesting NMD activation following SF3B1 silencing. We used Human Exon-Junction arrays (Affymetrix) to evaluate global transcript exon usage in the K562 and TF1 cell lines with SF3B1 knockdown. We observed significant differential exon usage in genes involved in RNA degradation, spliceosome, cell cycle and apoptosis. We further observed aberrant splicing of the candidate gene ABCB7 showing exon skipping and TP53 gene showing exon skipping as well as intron retention.We have investigated the changes in the transcriptome in CD34+ cells from MDS patients with SF3B1 mutation by RNA sequencing and found many genes showing significant differential exon usage including CCND1, EIF3B, FKBP1A, BCL2 and RB1. Using Ingenuity Pathway Analysis we identified alternative splicing pattern of genes involved in cell cycle, RNA processing, mTOR signalling and P53 signalling pathways.We have studied CD34+ cells from MDS patients with SF3B1 mutation in vitro and observed impairment in cell growth compared to CD34+ cells from healthy controls or from MDS patients without splicing mutations. In colony forming assays we observed a decrease in the number of erythroid or myeloid colonies derived from CD34+ cells of patients with SF3B1 mutation compared to patient CD34+ cells without splicing factor mutation. The identification of SF3B1 downstream targets in SF3B1 mutant and wild-type erythroid and myeloid colonies from MDS patients is in progress using RNA sequencing.Our results show that knockdown of SF3B1 in haematopoietic cell lines results in impaired cell growth, deregulated global gene expression and aberrant splicing. Studies of the haematopoietic progenitor CD34+ cells of patients with SF3B1 mutation show impaired cell growth and erythroid differentiation as well as deregulation of many pathways including the cell cycle and RNA processing. The identification of the key target genes affected by the common splicing mutations in MDS is critical to our understanding of how the mutations contribute to the pathogenesis of this disorder. Disclosures:Maciejewski:NIH: Research Funding; Aplastic anemia&MDS International Foundation: Research Funding.

Comprehensive Identification Of Germline Alterations In Telomerase Complex Genes By Whole Exome Sequencing Of MDS and Related Myeloid Neoplasms

In addition to classical familiar forms of bone marrow failure, some cases of aplastic anemia (AA) have been linked to inherited germ line polymorphism/mutations of telomerase machinery, leading to excessive telomere shortening. Germline telomere maintenance machinery mutations have been also been found in a proportion of acute myeloid leukemia (AML) and Myelodysplastic syndromes (MDS) patients (pts). However, the molecular pathogenesis of adult MDS and AML is complex and determination of genetic risk factors in addition to established familial and congenital syndromes has been difficult. To date targeted sequencing has been used for mutational screens with the inherent limitations of limited exome coverage, empiric bias and labor intensity. New generation (NGS) whole genome approaches prioritize somatic mutations as initial discovery targets, but the availability of sequenced cohorts allows also for detection of germline lesions both in a targeted and an unbiased fashion.Using NGS we studied 136 pts (mean age, 68.8 years, range 41-85) with MDS and related myeloid neoplasms for the presence of non-synonymous polymorphisms (SNV), which could affect telomerase machinery. These genes included TERT, DKC1, SMG6, NOP10, POT1, WRAP53, NHP2, GAR1, TINF2. No somatic defects of the telomerase complex were detected. We focused on novel sequence alterations or those described in available databases with a population allelic frequency of less than 5%. We identified 45 non-synonymous germline sequence alterations in 39 cases (32%). Most frequent SNV were found in TERT (n=15), DKC1 (n=7), SMG6 (n=6), NOP10 (n=4), POT1 (n=4), WRAP53 (n=4), while observations of NHP2 (n=3), GAR1 (n=1), TINF2 (n=1) were less prevalent. These variants were distributed in an almost mutually exclusive manner. Out of 3 variants in TERT, p.H412Y (n=3) and p.A279T (n=9) were reported to be pathogenic in bone marrow failure syndromes. In addition, p.A999T found in 8 cases in our cohort could also be pathogenic since it is less frequent in healthy controls. Similarly, p.441_442del (n=1), located in the N-terminal region, is a completely novel germline variant not detected in 6500 samples publicly available in ESP6500. In the pAML cohort (TCGA; n=197), the observations of germline variants for these telomerase complex genes were SMG6 (n=21), POT1 (n=19), NHP2 (n=1), NOP10 (n=1) GAR1 (n=1).Next, we analyzed clinical characteristics, including treatment responsiveness as assessed per modified 2006 IWG response criteria. The mean age of the 39 patients with germline telomerase machinery alterations was 67 years, 24% (9/39) were younger (age<60 years) compared to 12% (12/97) of wild type (WT; p=.12). Of note, 58% of these cases had a family history of solid tumors including breast, gastrointestinal and prostate and 8% (3/36) had a family history of myeloid malignancies. 41% (16/39) of the telomerase mutants had higher-risk MDS/sAML at presentation compared to 23% in WT cases (23/97; p=.19). A higher percentage of mutants also had complex cytogenetics compared to WT (35% vs. 13%; p=.01). Response rates to common therapies, including hypomethylating agents were similar, but we noted that none of the carrier cases (n=16) treated with lenalidomide showed therapeutic responses (0% vs. 37%). The mean overall survival of the carrier cases was lower compared to the WT (36 vs. 39 months, p=.10). When we studied cases with telomerase alterations for the presence of coinciding somatic mutations, using a targeted deep sequencing panel of the 100 most common mutations acquired in pts with germline telomerase complex alterations, we found most common the acquisition of DNMT3 (18% vs. 6%, p.10) and cohesin mutations (13% vs. 4%,p=.11).In sum, unbiased NGS sequencing approaches in MDS and related myeloid neoplasms allowed for identification of genetic germline alterations in telomerase maintenance machinery at higher rates than previously detected using targeted screening approaches, suggesting that such genetic defects may more frequently than previously thought contribute to cryptic and likely complex genetic predisposition to these diseases. Disclosures:Makishima:AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding.

Differences In Perceptions Of Disease and Treatment Effectiveness and Adherence Between Physicians and Patients With Myelodysplastic Syndromes (MDS)

Background and MethodsMDS are complex conditions, described with sometimes confusing terminology (e.g., “refractory anemia”), and contemporary drug therapies (tx) for MDS require repeated administration cycles to achieve clinical effect. Lack of disease understanding or premature tx discontinuation may result in poorer outcomes for patients (pts). To better understand physician (MD) and pt perceptions about MDS and tx decisions, we conducted two online surveys: one for pts with MDS and one for healthcare providers (HCP) registered with the non-profit Aplastic Anemia & MDS International Foundation. The protocol and consent were approved by a central Institutional Review Board. Pt and HCP surveys consisted of 57 and 49 questions, respectively, and assessed understanding of MDS, perceptions of specific tx, barriers to tx adherence, and overall tx experience. Data were analyzed using proportions, means, and medians; groups were compared using a Chi-squared test. ResultsOf 4,039 pts invited to participate via e-mail, 477 (12%) complete responses were received from 42 US States. Of responders, 247 (52%) were men; 63% were ≥age 60; pts were diagnosed with MDS a median of 5 years prior to the survey (range, 0-32 years). Of 4,594 HCPs invited to participate, 120 (3%) complete responses were received. Due to low participation among other HCP groups, only MD responses were examined. Of the 61 MDs (from 23 US states), 35 (57%) practice in an academic setting and 26 (43%) in the community setting. Survey responses from self-designated academic and community MDs did not differ significantly. Among MDs, 48% reported they see 5-19 new MDS pts per year. Only 10% of pts reported MDS was described to them as “cancer”, compared to how 59% of MDs stated they described it (p<.001). Only 29% of pts reported that MDS is “curable”, compared to 52% of MDs (p<.001). Forty-two percent of pts had received at least one disease-modifying tx: azacitidine (AZA, 58%), decitabine (DAC, 27%), lenalidomide (LEN, 35%) or hematopoetic stem cell transplant (HSCT, 26%) (total >100% due to multiple answers).MD and pt perceptions of active tx were significantly different, with MDs overestimating quality of life (QOL) benefits and underestimating the burden of tx on pt activities. [Table 1]MDs interpreted the benefit of active tx significantly higher than pts, however pts perceived the actual tx experience more positively than MDs. [Table 2]Most pts (81%) reported that MDs had the most influence on their tx decisions. Sixty-nine percent of MDs reported recommending stopping tx prior to the completion of tx regimen. Reported reasons diverged between pts and MDs, including burden of tx exceeding the benefit to the pt, as well as perceptions that the impact on the pt and family was too great. [Table 3]Table 1Active Tx – MD & Pt PerceptionsAZADACLENStatementsMDs n=55Pts n=115P-valueMDs n=23Pts n=54P-valueMDs n=40Pts n=69P-valueCan improve QOL89%63%P=.00185%65%NS80%56%P=.023Makes patients (me) feel better while taking it62%29%P<.00162%46%NS63%35%P=.009Has no side effects7%24%P=.0145%28%P=.0125%24%P=.019Makes regular activities difficult on tx days27%45%P=.03826%41%NS30%25%NSMade regular activities difficult for days following tx25%43%P=.04631%44%NS60%21%P<.001Table 2Tx for MDS – MD & Pt PerceptionsMD StatementsMDs N=61Pts n=200P-valuePt StatementsMy patients' lives are better because I prescribed tx95%79%P=.007My life is better because I received txMy patients are glad they have received tx93%82%P=.049I'm glad I had the txThe tx cured my patients' MDS44%25%P=.005The tx cured my MDSTx is uneventful16%34%P=.016Tx was uneventfulGetting tx is easy21%51%P<.001Getting tx was easyTable 3Barriers to Adherence – MD & Pt PerceptionsStatements (pt)MDs n=42Patients n=116p-valueBurden of tx outweighed benefit to pt (me)69%35%P<.001Burden of tx was too great on the family/caregiver50%23%P=.002Patient fatigue was too great to continue60%33%P=.004Tx made the patient (me) feel too sick to continue79%33%P<.001Tx side effects interfered with the pt's (my) regular activities74%35%P<.001 ConclusionPhysicians and pts with MDS have distinct views of the value of tx for MDS, with MDs underestimating the impact of tx on pt QOL while overestimating it as justification for stopping tx. Improved communication may improve understanding of disease and impact of active treatment and achieve better tx adeherence and responses. Disclosures:No relevant conflicts of interest to declare.

Aplastic Anemia & MDS International Foundation (AA&MDSIF): Bone marrow failure disease scientific symposium 2012

Aplastic anemia (AA), myelodysplastic syndrome (MDS), and paroxysmal nocturnal hemoglobinuria (PNH) are rare disorders of bone marrow failure. Once considered distinct entities, these three diseases are now believed to have overlapping pathophysiologies. The Aplastic Anemia and MDS International Foundation, a nonprofit organization that supports patients and families living with bone marrow failure disorders, sponsored a scientific symposium in Bethesda, MD, in March 2012. This report summarizes the symposium presentations by 30 of the world's leading AA, MDS, and PNH researchers on recent findings, current areas of controversy, and recommendations for basic and clinical research to advance the field.

Haploinsufficiency of Mir-146a in High-Risk Del(5q) MDS/AML Requires an Intrachromosomal Gene Network Involving p62/TRAF6/NF-κB

AbstractAbstract 557Deletion of chromosome 5q (del(5q)) is one of the most common cytogenetic abnormalities in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Commonly deleted regions (CDR) have been mapped to 5q31.1 and 5q33.1 in del(5q) AML and MDS, respectively. Although there has been extensive efforts to identify candidate genes within the CDRs and decipher which genes contribute to the high-risk versus low-risk phenotypes, more recent findings indicate that deletions involving the telomeric bands of 5q are associated with more aggressive forms of del(5q) MDS/AML (Jerez et al., JCO, 2012). To determine which genes on the telomeric extremes of chr 5q contribute to the aggressive phenotype associated with these patients, we performed a gene expression and functional analysis of candidate tumor suppressor (TS) genes located between ∼5q34 to 5q35.3. Among the 7 candidate TS genes localized on the telomeric extreme, expression of miR-146a is significantly reduced in del(5q) MDS/AML marrow cells with extended deletions as compared to ones with shorter deletions. In addition, low expression of miR-146a correlates with increased marrow blasts and shorter overall survival of patients with del(5q). To investigate the effects of miR-146a on hematopoietic stem/progenitor cell (HSPC) function, we stably knocked down miR-146a in human CD34+ cord blood and mouse HSPC-enriched cells. HSPC cells with reduced miR-146a exhibited features of malignant transformation, including increased survival, proliferation, and colony replating in methylcellulose. The mechanism underlying reduced miR-146a expression was investigated by performing gene expression profiling on marrow cells from MDS/AML patients with extended and short del(5q). Gene Ontology and Gene Set Enrichment Analysis revealed that patients with low expression of miR-146a exhibited a significant increase in cell cycle, immune response, and NF-kB target genes. These findings are consistent with the model that reduced levels of miR-146a results in derepression of TRAF6, a signaling protein within the innate immune pathway and activator of NF-kB (Starczynowski et al., Nature Medicine 2010). TRAF6 forms a signaling complex with Sequestosome 1 (SQSTM1/p62), a gene located within the telomeric deleted region in del(5q) on band 5q34. Surprisingly, expression of p62 was not reduced, but rather overexpressed in del(5q) MDS/AML patients with the extended deletions and inversely correlated with miR-146a expression. The compensatory expression of p62 is due in part by loss of miR-146a. RNAi-mediated knockdown revealed that p62 and TRAF6 are essential for NF-kB activation and survival of human MDS/AML cell lines with low miR-146a expression. In summary, we identified that miR-146a is a TS-like gene in MDS/AML patients with extended deletions of chr 5q, and that reduced miR-146a expression increases HSPC survival/proliferation and NF-kB signaling via p62 and TRAF6. Therefore, we propose that inhibiting the p62/TRAF6/NFκB intrachromosomal gene network represents a novel targeting strategy in high-risk MDS/AML patients with extended chr 5q deletions. Disclosures:Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.

STAT3-Mutations Indicate the Presence of Subclinical Self-Reactive Cytotoxic T Cell Clones in Aplastic Anemia and Myelodysplastic Syndromes

AbstractAbstract 646Large granular lymphocyte leukemia (LGL) is often associated with immune cytopenias, but can also occur in the context of myelodysplastic syndrome (MDS). LGL shares certain pathogenetic similarities with aplastic anemia (AA) and some forms of MDS, in which cytopenias are related to immune suppression of normal hematopoiesis. In these conditions, the inhibition of hematopoietic progenitor and stem cells has been described to be mediated by mostly polyclonal cytotoxic T lymphocytes (CTL). Previously, using molecular analysis of TCR VB repertoire in these diseases we have demonstrated oligoclonal skewing clonal of CTL spectrum that was reminiscent (albeit less pronounced) to that seen in LGL. These observations support the theory that these CTL expansions correspond to a cellular reaction against autologous hematopoietic targets. Detection of STAT3 mutations would substantiate the hypothesis that autoimmune reactions can be due to intrisic genetic lesions in autoimmune cells.The recent discovery of recurrent somatic STAT3 mutations appears to be the key molecular lesion promoting clonal outgrowth of autonomous CTL clones in LGL. This finding raised the hypothesis of whether those mutations could be found in other bone marrow failure (BMF) states and whether they could be diagnostically useful and associated with distinct clinical features. Initially, we have directly sequenced STAT3 exons in 120 T-LGL cases and identified 33 mutations in 32 cases (27%). All mutations were located in the domain of STAT3 (residues 585–688) that shares homology with Src homology 2 (SH2) domains. The STAT3 SH2 domain mediates STAT3 dimerization via binding of phosphotyrosine residue Y705. Two mutations, Y640F and D661Y, accounted for 80% of the somatic variations found, enabling the design of a more sensitive ARMS-PCR method for each of these alterations, suitable for the massive screening we have envisioned for AA and MDS.In BMF, we have first identified 21 MDS patients with known LGL and screened them for the presence of STAT3 mutations: in the CTLs of 6/21 of these patients STAT3 mutations were found and thus less clinically apparent LGL expansion could also be present in more classical MDS. Thus, we extended our screening to CTLs from an additional 368 patients with MDS and no suspected concomitant LGL: we identified 9 additional patients with STAT3 mutated clones. MDS patients carrying STAT3 mutant CTL clones had both advanced and low risk disease (low, n=1; int-1, n=4; int-2, n=8; High, n=2). These patients were characterized by a higher frequency of hypocellular bone marrow (55 vs. 10.5%; p<.001), and neutropenia at diagnosis (p<.04). No significant differences were found in overall survival. By analogy we also searched for STAT3 CTL clones in AA (N=148) and PNH (N=30). In total, we have identified 17 (10%) AA patients with STAT3 mutant CTL clones: all of these patients did not display manifest LGL. Clinically, these patients had a higher proportion of non severe AA (40% vs. 23%) and were more likely to respond to first line immunosuppression (76 vs., 65%) though no statistical significance was reached. In addition, cases with BMF and a subclinical mutant CTL clones were retrospectively tested for the presence of a TCR rearrangement: an oligoclonal (40% of cases) or monoclonal pattern (20% of cases) was seen in most of patients. In sum, the surprising discovery of STAT3 mutated clones in BMF states seems to be predominantly in AA patients and can be also found MDS cases with mainly, hypoplastic features, suggesting that STAT3 mutated self reactive CTL clones may play a role in immune pathogenesis of these conditions. Disclosures:Koskela:Novartis: Honoraria; BMS: Honoraria; Janssen-Cilag: Honoraria. Mufti:Celgene: Consultancy, Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria. Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.

Impact of Mutations in the Spliceosome Machinery and Ring Sideroblasts in Patients with Myeloid Malignancies Who Received Conventional Chemotherapy or Allogeneic Hematopoietic Cell Transplantation

AbstractAbstract 1973Mutations in the spliceosome machinery have recently been identified in myeloid neoplasms including acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), and MDS/MPN overlap syndromes. Somatic alterations in SF3B1, U2AF1 and SRSF2 are the most frequently affected spliceosome genes in myeloid malignancies with the frequencies of any of the 3 genes being 39% in low risk MDS, 11% in high risk MDS/AML (primarily U2AF1), and 24% in MDS/MPN, (primarily SRSF2). While SF3B1 mutations as well as ring sideroblasts (RS) appear to be associated with improved survival and lower risk of leukemic evolution, mutations of U2AF1 and SRSF2 appear to be associated with poorer outcomes. Allogeneic hematopoietic cell transplantation (HCT) is the only curative option for many myeloid malignancies, however, the prognostic role of SF3B1, U2AF1 or SRSF2 mutations in patients (pts) undergoing high intensity chemotherapy (HIC) and HCT has yet to be investigated.We evaluated 404 pts with the diagnosis of AML (n=96), MDS and MDS/MPN (n=294), MPN (n=13), and congenital sideroblastic anemia (n=1). 35 pts received HIC defined as treatment with 7+3, high dose cytarabine, clofarabine, or other comparable chemotherapy in the context of a clinical trial. 97 pts underwent a myeloablative (n=69) or reduced intensity (n=28) allogeneic HCT from an HLA matched related (n=39), unrelated (n=50) or cord blood (n=8) donor from April 2003 until August 2011. Median age of the whole cohort was 69 and those who underwent BMT at time of transplant was 52 (range 19–70). We performed direct sequencing for the three most commonly mutated spliceosome genes, SF3B1 (exon 13–16), SRSF2 (exon 1 and 2) and U2AF1 (exon 2–6). Given the close association between SF3B1 mutations and RS, we analyzed the impact of the presence or absence of RS on the outcomes associated with the type of therapy received. We found a total of 24 SF3B1 mutations in MDS and MDS/MPN (primarily in pts with RS), 37 SRSF2 mutations in MDS and MDS/MPN (primarily CMML), and 13 U2AF1 mutations in MDS, MDS/MPN and secondary AML.Among pts with MDS and MDS/MPN, significant overall survival (OS) differences were found between SF3B1 mutant and wild type (WT), (83 vs. 31 months, p=0.001) and U2AF1 or SRSF2 mutant (18 vs. 37 months, p=0.007). The U2AF1 mutation by itself was more strongly associated with poor outcome (8 vs. 37 months, p<0.0001), compared to an SRSF2 mutation (26 vs. 36 months, p=0.16). Analysis of survival according to treatment type revealed that no pts who underwent HIC or HCT carried an SF3B1 mutation. The presence of RS was also associated with improved survival (38 vs. 25 months, p=0.04), and this benefit remained if pts received HIC (18 vs. 9 months, p=0.04). However, this benefit became non-significant if pts underwent HCT. There were 19 pts who had either an U2AF1 or SRSF2 mutation that underwent HIC. Although it appeared that having either mutation was associated with worse survival, (9 vs. 21 months), it did not reach statistical significance (p=0.46). Among pts who underwent HCT, there were 10 pts who carried either an U2AF1 or SRSF2 mutation. Those pts with mutations appeared to have superior survival with HCT compared to WT, (53 vs. 34 months), but this did not reach statistical significance (p=0.54). Among pts with either an U2AF1 or SRSF2 mutation, those who underwent HCT had a superior survival (53 vs. 17 months, p=0.05) compared to those who received conventional chemotherapy, defined as HIC or low intensity chemotherapy (LIC), such as hypomethylating agents. There was no difference in survival between receiving HCT or conventional chemotherapy in pts who were WT for U2AF1 or SRSF2 (34 vs. 31 months, p=0.08).Consistent with previous findings of improved survival and loss of SF3B1 with leukemic progression in MDS and MDS/MPN pts, we found no SF3B1 mutations in our cohort of pts receiving HIC or HCT. In contrast, we found poorer outcomes with SRSF2 or U2AF1 mutations. Although the incidence of these mutations was rare in our cohort of pts, we found statistically significant survival benefit for pts with these mutations if they underwent HCT compared to conventional chemotherapy. This is the first study evaluating the impact of spliceosomal mutations on the survival outcomes of pts undergoing HCT. Our preliminary findings of improved survival with HCT in poorer-risk spliceosome mutations is provoking and further molecular analysis is ongoing. [Display omitted] Disclosures:Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.

Various Germline Congenital Disorder Genes Are Somatically Mutated in Myeloid Malignancies

AbstractAbstract 1405Genes involved in congenital genetic cancer susceptibility syndromes are also targets of somatic mutations in various tumors. Examples include WT1, NF1, CBL, TP53 and MLL2 affected both in germ line as well as somatic mutations present in malignant disorders. To apply this approach to investigation of pathogenic mutations in myeloid malignancies, we selected 183 congenital disorders in which germline mutations of disease specific genes are reported to be pathogenic. Their main clinical presentations are skeletal abnormalities (N=54 disorders), skin abnormality (N=24), mental retardation (N=17) and hematological disorders (N=12). In total, we searched for mutations in 204 genes associated with these congenital disorders.We analyzed whole exome of various myeloid malignancies, including 60 cases with myelodysplastic syndromes (MDS), 29 MDS/MPN, 5 with MPN and 122 with acute myeloid leukemia (AML) and found somatic mutations in 62 genes, which also mutated in germ line in various congenital syndromes. Of those, the most frequently mutated genes were TP53 (25 cases) and WT1 (16 cases), associated with germline mutation of Li-Fraumeni syndrome and Wilms tumor, respectively. Some somatic mutations, for example, NF1 (R1276Q) and PTPN11 (D61N), were exactly the same as observed in corresponding congenital disorders (Neurofibromatosis or Noonan syndrome). One of the novel findings is that somatic SET binding protein 1 (SETBP1) mutations (D868N, G870S and I871T) were commonly observed in sAML and CMML, and were identical to germline mutations in Schinzel-Giedion syndrome (see designated abstract). We found recurrent somatic SETBP1 mutations in 15% of each CMML and sAML. Moreover, multiple genes pathogenic in Usher syndrome (congenital hearing and vision loss, complicated by vasoproliferative retinal tumor), were somatically mutated in various myeloid neoplasms. Out of 9 genes which are causative for this syndrome, 15 mutations of 6 genes (MYO7A, USH1C, CDH23, PCDH15, USH2A, and GPR98) were observed in 13 cases, including 2 frameshift and 13 missense mutations. These genes coordinate with each other to form a functional network. CDH23 and PCDH15 are cadherins and act as cell adhesion molecules. MYO7A are actin-based motor molecules with a variety of functions. USH1C serves as an anchor and codes for a scaffolding protein to form a complex with all the other proteins. Through the PDZ binding site, USH1C forms a complex with CDH23, which was the most frequently mutated gene in this family (1 frame shift and 3 misssense mutations). CDH23 mutations were observed in 2 cases with primary AML, sAML and MDS. Specifically, a somatic missense mutation G2771S of CDH23 in a secondary AML case was identical to germline of Usher syndrome. The second most frequently mutated gene, GPR98, is located in 5q14.3 locus; a small hemizygous clone found in del5q of an MDS case. In a serial sample analysis, this mutation increased to become the larger main clone during AML evolution. Moreover, in this case, an additional CDH23 mutation was acquired in the course of leukemic expansion. In such cases with Usher syndrome gene mutations, U2AF1, ZRSR2, EZH2, IDH2 and ETV6 mutations were also observed, suggesting pathogenic cooperation with these well-known tumor suppressor genes and oncogenes. Disclosures:Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding. Makishima:Scott Hamilton CARES Initiative: Research Funding.

Novel Recurrent Mutations in the Ras-Like GTP-Binding Gene Rit1 in Myeloid Malignancies

AbstractAbstract 558In addition to chromosomal and epigenetic abnormalities, somatic mutations constitute key pathogenic lesions in myeloid neoplasms. Individual somatic mutations or various combinations may be both valuable prognostic markers and targets for new rational therapies. Among them, RAS family genes are ubiquitous oncogenes associated with various cancers. Recurrent canonical mutations in the nucleotide binding domains in NRAS and KRAS result in constitutively activated proteins. In myeloid neoplasms, RAS mutations convey a poor prognosis and are often found in acute myeloid leukemia (AML), myelodysplastic syndromes (MDS) and, rarely, myeloproliferative neoplasms (MPN).We applied whole exome sequencing to paired germline vs. leukemia samples in 65 cases of MDS, 36 MDS/MPN and 32 sAML. We focused our study on the RAS protein superfamily of small GTPases and identified mutations in 3% and 6% of KRAS and NRAS, respectively. Most significantly, we identified somatic recurrent mutations in the F82 residue of Ras-like without CAAX1 (RIT1) gene in 2 patients with chronic myelomonocytic leukemia (CMML) and secondary AML (sAML), respectively. We confirmed the somatic nature of both mutations in sorted CD3+ cells from each patient (pt).RIT1 gene encodes a member of Ras-related GTPases, involved in the p38 MAPK and AKT signaling pathway that mediates cellular survival in response to stress. RIT1 gene amplification has been found in 26% of hepatocellular carcinoma. However, neither amplification nor mutations of this gene have been reported in myeloid malignancies. We thus focused this line of experimentation on this somatic mutation. To establish clinical associations we further studied a cohort of 322 patients with various myeloid malignancies by Sanger sequencing and detected somatic RIT1 mutations in an additional 6 (2%) cases. All mutations were located in exon 5, in the 81 and 82 residues, which encode the switch II domain of this protein, an effector region very close to the GTP-binding site G3, and which is highly conserved among species. Among the 8 mutant cases, 5 (63%) pts had CMML, resulting in a higher frequency of mutations in this subcohort of pts (5 out of 57 CMML, 9%). The other 3 mutations were found in one primary (p)AML (M5b subtype) (1 out of 58 pAML, 2%) and two high-grade MDS, one refractory anemia with excess blasts (RAEB)-2 and one sAML(RAEB-T in the FAB-classification) (2 out of 80, 2.5%).RIT1 mutations were heterozygous in all cases except for one case with trisomy 1 and duplication of the mutant allele. In the cases of WES, we estimated an allelic frequency of ∼35%, consistent with the presence of a heterozygous mutation in ∼70% of sample cells. Because of the large size of the clone and serial samples showing RIT1 mutation since the time of initial diagnosis, it is likely that RIT1 may be of ancestral origin.As RAS-family gene amplifications have been described in cancer, we also studied the presence of amplifications of the RIT1 locus (1q22) by SNP-A. We found 10 cases characterized by a gain involving the RIT1 region (1q21.1-q44): 4 (40%) cases had a diagnosis of CMML, 4 (40%) had myelofibrosis, whereas the remaining patients had MDS (one RAEB-1 and a RA). Quantitative RT-PCR showed RIT1 overexpression in mutants and in patients with 1q amplification (median normalized relative ratio 0,51 and 0,40, respectively) compared to patients with wild type RIT1 and no amplification in 1q (median normalized relative ratio 0,15; P=.039).We theorized that activating RIT1 mutations may constitute a suitable therapeutic target. Because AKT inhibitors can block AKT phosphorylation and therefore reverse the antiapoptotic effect of mutant RIT1, we tested whether AKT inhibitor V (Triciribine) can selectively abrogate the growth of primary cells with RIT1 mutation. In in vitro suspension cultures, a 65% of reduction proliferation was observed with significant effects even at 0.1μM concentrations.In sum, somatic recurrent RIT1 mutations are novel lesions involved in the molecular pathogenesis of myeloid cancers, presumably early in the development of the disease. Moreover, amplifications of RIT1 also lead to overexpression of this Ras-like GTP-ase. Specifically, these abnormalities appear to be more frequent in patients with CMML, but also can be found in other types of MDS. Disclosures:Makishima:Scott Hamilton CARES Initiative: Research Funding. Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.

Non-Cytotoxic Differentiation Therapy Based On Mechanism of Disease Produces Complete Remission in Myelodysplastic Syndromes (MDS) with High Risk Cytogenetics

AbstractAbstract 1696The standard treatment concept and approach in MDS aims for apoptosis-induction in expanding malignant clones in the hope that this will permit recovery by functionally normal hematopoietic stem cells (HSC). This concept is fundamentally undermined, however, by frequent genetic deletion in MDS/AML cells of key apoptosis genes. Hence, therapy selects for relapse with the most apoptosis-resistant malignant subclones (e.g., p53-null) while destroying crucial normal HSC (which have intact apoptosis-pathways). Observations regarding biology of sustained proliferation (MYC activity) in MDS/AML suggested an alternative approach: key late-differentiation MYC-antagonist genes (e.g., CEBPE) are repressed epigenetically even though lineage-specifying transcription factors (TF) (e.g., CEBPA) that drive expression of these genes are highly expressed. The basis for this paradox has been exposed: MDS/AML associated genetic abnormalities (e.g., RUNX1 mutation) favor recruitment of corepressors (e.g., DNMT1) instead of coactivators to lineage-specifying TF, thereby repressing differentiation target genes. Thus, in pre-clinical MDS/AML models, depleting DNMT1 by non-cytotoxic methods triggers MYC-antagonist expression and irreversible cell cycle exit by p53/p16-independent differentiation pathways (CEBPE-p27). The same treatment spares normal HSC, which unlike AML stem cells, do not express high levels of lineage-specifying TF.To translate these observations, this NIH-supported clinical trial (NCT01165996) incorporated the following novelty: (i) Lower dose of the cytidine analogue decitabine (DAC, Eisai) than in any previous trials (3.5–7 mg/m2) since hemoglobinopathy clinical trials demonstrated that these low doses deplete DNMT1 in normal HSC without cytotoxicity/apoptosis; (ii) SC instead of IV administration to avoid high peak levels that cause apoptosis; (iii) Use of the lack of toxicity to administer drug frequently 1–3X/week, to catch more MDS cells in S-phase via greater exposure times and distribution than in previous MDS trials (FDA-approved schedules only treat MDS cells entering S-phase in the first few days of multi-week cycles); (iv) Scientific correlates to measure non-cytotoxic, epigenetic-differentiation mechanism of action.Sample size = 25 as planned, 10/25 (40%) were relapsed/refractory after 5-azacytidine and/or lenalidomide, median age = 72y (range 46–85y), median disease duration = 900d (range 60–4680d). Anti-emetics were not needed, and cytotoxic side-effects e.g., hair loss did not occur. Neutropenic fever (NF) occurred in 11 subjects (5 had NF and 8 had ANC<500 prior to therapy). There was one treatment-associated fatality from sepsis. Though non-cytotoxic, suppression of clonal hematopoiesis is intended: nadir preceded complete remission (CR) 4/25 (16%) or hematologic improvement (HI) 7/25 (28%) (CR + HI = 47%) and 10/25 stable disease (SD) (CR+HI+SD = 84%) (IWG criteria). In transfusion dependent patients durations of transfusion independence for platelets (n=5) were median 343d (range 186–707d), 3/5 ongoing; for RBC (n=10) median 399d (range 290–696d), 5/10 ongoing. Complete cytogenetic remissions (CyR) occurred in 6/12 (50%) and partial CyR in 2/12 (17%) (overall CyR = 67%). Consistent with a p53-independent mechanism, CyR occurred even in poor risk cytogenetics, and increasing DAC frequency salvaged loss of response. Higher marrow cellularity was the best predictor of CR/HI, and CyR was substantially higher than CR/HI, underscoring that despite efficacious clone suppression, marrow HSC reserve is crucial to relief of cytopenia. Non-cytotoxic epigenetic-differentiation mechanism was confirmed by marrow IHC and FCM measurement of DNMT1, g-H2AX, p27/CDKN1B, MYC, and KI67 (3rd party quantification).Differentiation therapy transformed the outlook for M3 AML. Not surprisingly, a worthy goal has been to extend this paradigm. Biology of sustained MYC activity in MDS/AML guided logical repurposing of DAC for non-cytotoxic differentiation therapy even in MDS/AML with complex cytogenetics. Tolerance permits ready application despite age or comorbidities. Treatment exposure time and surrogates of normal HSC reserve are more important predictors of CR/HI than disease-mutations, and some patients may require separate measures (e.g., cytokines) to boost normal HSC diminished by age and previous insults. Disclosures:Reu:Celgene: Research Funding. Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding. Saunthararajah:Cleveland Clinic Innovation: patent application for oral THU-decitabine., patent application for oral THU-decitabine. Patents & Royalties. Off Label Use: lower dose of decitabine, subcutaneous administration, metronomic administration.

Disparity in Perceptions of Disease, Treatment Effectiveness and Treatment Adherence Between Physicians and Patients with Myelodysplastic Syndromes (MDS)

AbstractAbstract 4949 Background and Methods:Previous studies have suggested that many patients (pts) with MDS have incomplete understanding of their disease and its treatment. In addition, contemporary drug therapies (tx) for MDS often require repeated treatment administration cycles to achieve clinical effect, and premature discontinuation or incomplete adherence may result in lack of benefit in pts who might have responded to a longer course of tx. In order to better understand physician and pt perceptions about MDS and decisions about continuing tx, we conducted two online surveys in February 2012: one for pts with MDS and one for healthcare providers (HCP) registered with the Aplastic Anemia & MDS International Foundation. A $50 Amazon gift card was offered to the first 200 respondents in each group; acceptance constituted consent. The protocol was approved by a central Institutional Review Board. Pt and HCP surveys consisted of 57 and 49 questions, respectively, assessing understanding of MDS, perceptions of specific tx, perceived barriers to tx adherence, and overall tx experience. Data were analyzed using proportions, means and medians, and comparisons between groups were calculated using Pearson’s Chi square where appropriate. Results:477 complete pt responses were received from 42 US States, as well as 120 complete HCP responses. Of pt responders, 247 (52%) were men, 63% were ≥60 years old, and the median time from MDS diagnosis was 5 years (range, 0–32 years). Because of low participation among other HCP groups, only physician (MD) responses are analyzed here. Of the 61 physicians (from 23 US states), 35 (57%) practice in an academic setting and 26 (43%) in the community setting; 48% reported they see 5–19 new MDS pts per year. Survey responses from academic and community physicians did not differ significantly.Only 10% of pts reported agreement that MDS is a “cancer”, compared to 59% of physicians (p<. 001). Only 29% of pts report that MDS is a “curable” condition, compared to 52% of physicians (p<. 001). Forty-two percent of patients had received at least one active tx: azacitidine (AZA, 58%), decitabine (DAC, 27%), lenalidomide (LEN, 35%) or hematopoetic stem cell transplant (HSCT, 26%) (total >100% due to multiple answers). Physicians and pt perceptions of specific active tx were significantly different, especially regarding quality of life (QOL), adverse events and impact of tx on pt activities. [Table 1]. Physicians viewed the potential benefits of active tx significantly greater than did pts, however pts perceived the actual tx experience more positively than doctors. [Table 2]Table 1Specific Tx – MD & Pt PerceptionsAZADACLENStatementsMDs n=55Pts n=115P-valueMDs n=23Pts n=54P-valueMDs n=40Pts n=69P-valueCan improve QOL89%63%P=.00185%65%NS80%56%P=.023Makes patients (me) feel better while taking it62%29%P<.00162%46%NS63%35%P=.009Has no side effects7%24%P=.0145%28%P=.0125%24%P=.019Makes regular activities difficult on tx days27%45%P=.03826%41%NS30%25%NSMade regular activities difficult for days following tx25%43%P=.04631%44%NS60%21%P<.001Table 2Tx for MDS – MD & Pt PerceptionsMD StatementsMDs N=61Pts n=200P-valuePt StatementsMy patients' lives are better because I prescribed tx95%79%P=.007My life is better because I received txMy patients are glad they have received tx93%82%P=.049I'm glad I had the txTx cured my patients' MDS44%25%P=.005The tx cured my MDSTx is uneventful16%34%P=.016Tx was uneventfulGetting tx is “easy”21%51%P<.001Getting tx was “easy”Sixty-nine percent of physicians reported recommending stopping tx prior to the completion of a planned tx course, most commonly due to adverse events and the burden of therapy outweighing benefit. [Table 3]Table 3:Barriers to Adherence/Reasons for Discontinuation – MD & Pt PerceptionsStatements (pt)MDs n=42Pts n=116P-valueBurden of tx outweighed benefit to pt (me)69%35%P<.001Burden of tx was too great on the family/caregiver50%23%P=.002Patient fatigue was too great to continue60%33%P=.004Tx made the patient (me) feel too sick to continue79%33%P<.001Tx side effects interfered with the pt's (my) regular activities74%35%P<.001 Conclusion:Physicians and pts with MDS have disparate views of MDS characteristics and the value and limitations of tx for MDS. Improved communication and education may increase understanding of disease and achieve better results, including greater treatment adherence. Disclosures:Huber:Celgene: Research Funding, This study was funded by an unrestricted grant from Celgene to the AA&MDS IF. Other. Dennison:Celgene: Research Funding.

Reduced TET2 expression in Patients with Myelodysplastic Syndromes and Acute Myeloid Leukemia.

AbstractAbstract 2822 Introduction:Acquired mutations in TET2 and DNMT3A have been found in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), and may predict a worse survival in these diseases. TET2 mutations are considered to be a loss-of-function mutation and results in decreased 5-hydroxymethylcitosine (5-hmc) levels. In normal CD34+ cells, TET2 silencing skews progenitor differentiation towards the granulomonocytic lineage at the expense of lymphoid and erythroid lineages. Dnmt3a participates in the epigenetic silencing of hematopoietic stem cell regulatory genes, enabling efficient differentiation. Here, we attempted to evaluate the expression of TET2 and DNMT3A in total bone marrow cells from normal donors, patients with MDS and AML, and in CD34+ cells from MDS and normal controls during erythroid differentiation. Materials and Methods:The study included normal donors (n = 21), patients with MDS (n = 43) and AML (n = 42) at diagnosis. All normal donors and patients provided informed written consent and the study was approved by the ethics committee of the Institution. MDS patients were stratified into low and high-risk according to WHO classification (RCUD/RCMD/RARS=31 and RAEB1/RAEB2=12). TET2 and DNMT3A mRNA expression was assessed by quantitative PCR. CD34+ cells from normal donors (n = 9) and low-risk MDS patients (n = 7) were submitted to erythroid differentiation. Cells were collected and submitted to immunophenotyping for GPA and CD71 (days 6 and 12) and q-PCR for TET2 and DNMT3A expression (days 6, 8 and 12). Results of gene expression in normal donors and patients are presented as median, minimum-maximum, and were compared using Mann-Whitney test. Student t test was used for comparison of gene expression during CD34+ erythroid diferentiation. Overall survival was defined from the time of sampling to the date of death or last seen. Univariate analysis for overall survival was conducted with the Cox proportional hazards model. Results:TET2 expression was significantly reduced in both AML (0.62; 0.01–32.69) and MDS (1.46; 0.17–21.30) compared to normal donors (2.72; 0.43–31.49); P<0.0001 and P=0.01, respectively. TET2 expression was also significantly reduced in AML compared to MDS (P=0.0007). MDS patients were stratified into low and high-risk disease, and we still observed a significant reduction in TET2 expression in high-risk (0.73, 0.17–7.25) when compared to low-risk (1.58; 0.48–21.30; P=0.02) patients, but no difference was noted between normal donors vs. low-risk MDS, and high-risk MDS vs. AML. In MDS cohort, the median overall survival was 14 months (range 1–83), increased TET2 expression was associated with a longer survival (HR, 0.44; 95% CI, 0.21–0.91, P=0.03), and, as expected, WHO high-risk disease was associated with a shorter survival (HR, 10.16; 95% CI, 3.06–33.72, P<0.001), even though the confidence interval (CI) was large. TET2 expression did not impact survival in our cohort of AML patients. The erythroid differentiation was effective in cells from normal donors and MDS patients, as demonstrated by the flow cytometry analyses of GPA and CD71. TET2 expression was significantly increased on day 12 of erythroid differentiation, P<0.05. On the other hand, DNMT3A expression was similar between normal donors (0.74; 0.22–1.53), MDS (0.78; 0.26–3.46) and AML (0.95, 0.15–6.46), and during erythroid differentiation, with no impact on survival. Conclusion:These data suggest that decreased TET2 expression may participate in leukemogenesis, and supports the participation of TET2 in the erythroid differentiation of MDS. DNMT3A was not differentially expressed in AML and MDS, indicating that the presence of mutations in this gene may be the predominant mechanism of changes in protein function. We thus suggest that decreased TET2 expression may explain the reduced levels of 5-hmc found in TET2 wild type patients, and may become a predictive marker for outcomes in MDS and other myeloid diseases. Further studies would be necessary to better elucidate the clinical relevance and biologic significance of our findings, and whether the decreased TET2 expression results in hypermethylation in these diseases. Disclosures:Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.

Mutation Screening Associated with Chromosome 7 Abnormalities Using Next Generation Whole Exome Sequencing

AbstractAbstract 173One of the most common karyotypic abnormalities identified in myelodysplastic syndromes (MDS) is monosomy 7 (del7) or deletion of the long arm of chromosome 7 (del7q). The presence of del7/del7q carries a poor prognosis in MDS, MDS/myeloproliferative neoplasms (MPN) and acute myeloid leukemia (AML); the impact of these defects appears similar. Recently, a copy-neutral type of loss of heterozygozity (LOH also referred to as a somatic UPD) has been identified on 7q. Microdeletion on 7q corresponding to the EZH2 locus led to identification of inactivating mutations in this gene, though hemizygous EZH2 mutations are only rarely found and do not fully explain del7/7q pathogenesis. We performed a comprehensive analysis of myeloid neoplasms (N=189), using next generation whole exome sequencing technology, including MDS (N=34), MDS/MPN (N=26) or MPN (N=4) and 124 with AML (both primary and secondary). Among them, LOH7, involving del7/del7q were observed in 17% of cases (N=33). To minimize false positives and focus on the most prevalent/relevant somatic events, we implemented a rational bioanalyitic filtering approach, whereby paired DNA (tumor/CD3 lymphocyte) were sequenced and results aligned using Burrows-Wheeler Aligner and variants detected using GATK pipeline (Best Practice Variant Detection from Broad Institute). We focused on searching for del7/7q linked somatic mutational events involved comparisons of mutations in the area of del7q to cases diploid for this locus. We hypothesized that there may be heterozygous mutations of 7q, which could lead to functional haploinsufficiency that is also a result of del7q (haploinsuffcient theory, heterozygous mutations). Conversely, mutations may be either unique to del7q hemizygous inactivation, or shared between 7q diploid and haploid cases.In total, we found alterations in 12 genes located on chromosome 7 (6% of all alterations found). Using filtering strategies we narrowed the focus to “tier 1” mutations to avoid false positives; 11 mutated genes were found in cases with del7/7q and 2 in UPD7q. For example, novel hemzygous (but not heterozygous mutations) of an E3 ubiquitin ligase CUL1 gene were detected only in cases with del7/7q, suggesting that the wild type allele is protective. In cases with diploid 7q, 24 heterozygous alterations were observed (10 genes shared with del7/7q). The previously described EZH2 mutations were seen in heterozygous, homozygous and hemizygous configurations, but were most common in UPD7q (100%), while only 7% of del7/7q cases were positive. Notably, 5/12 mutant genes were located in commonly deleted regions (CDRs) either 7q22, 7q34 or 7q35–36. These CDRs also contain recurrently mutated lesions, including 7q22 (CUX1:n=4; STAG3:n=2), 7q34 (a splicing factor; LUC7L2: n=3) and 7q35–36 (EZH2: n=10). When we investigated the association between haploinsufficiency and heterozygous mutations, among those on del7/7q, cases with wild type forms of corresponding genes showed decreased expression. Similarly, such mutations were occasionally present in diploid configuration; here again the wild type cases showed a decreased expression. These findings suggest that mutated genes located in CDRs can be pathogenic due to both haploinsufficiency of WT genes and heterozygous mutations. EZH2 is a good example of such a gene. We also searched accessory genetic events observed on other chromosomes along with del7/7q and UPD7. By SNP-A, there were clear differences among 3 LOH7 groups, in which del7 was more associated with accessory chromosomal defects than cases with UPD7q or del7. Similarly, mutational patterns were specific to each LOH cohort. For example, while well known frequently mutated genes, such as U2AF1, TET2 and TP53, were commonly found in all 3 LOH7 groups, some specific genes, including the CSMD family, were uniquely observed in monosomy 7, not in del7q or UPD7. Similarly, LOH7q was associated with somatic mutations in SETBP1 and RUNX1. In conclusion, we detected several candidate genes that could be associated with del7/7q and UPD7. Some mutations were heterozygous in cases with diploid 7q and correlated with CDRs on del7/7q without mutation. Certain mutations are specifically observed with del7, while others are commonly observed in all categories of LOH7, including EZH2. Moreover, some genes outside of the chromosome 7 were coincidently mutated with LOH7. Disclosures:Makishima:Scott Hamilton CARES Initiative: Research Funding. Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.

Whole Exome Sequencing to Predict Response to Hypomethylating Agents in MDS

AbstractAbstract 1698Hypomethylating agents decitabine and azacitidine are standard treatments for myelodysplastic syndromes (MDS). In their use, one hopes to rectify cytopenias and prolong survival by retarding further disease progression. However, individual treatment responses vary from complete remission (CR) to complete refractoriness. In general, at least 4 cycles of therapy are administered prior to assessing response. Thus, patients may have prolonged exposure to ineffective therapy, suffering toxicities without clinical benefit, while alternative and potentially more effective treatments are delayed. Currently, there are no reliable phenotypic or mutational markers for predicting response to hypomethylating agents.Once whole exome sequencing (WES) became available for more routine analysis, we theorized that somatic mutational patterns may help identify patients who would most benefit from these drugs, thereby maximizing response rate by rational patient selection. To pursue this hypothesis, we screened a cohort of 168 patients with MDS who received either azacitidine or decitabine for the presence of somatic mutations. Only those who received sufficient therapy, i.e., completed at least 4 cycles, were selected for outcome analysis. Targeted Sanger sequencing, including a panel of up to 19 genes frequently affected by somatic mutations was performed. For a representative subset of 26 patients (this subset is expanding) of whom there were 15 responders and 11 non-responders, mutational analysis was performed by WES to select target genes for further analysis. WES utilizes paired DNA (tumor vs. CD3+ lymphocytes) to produce raw sequence reads aligned using Burrows-Wheeler Aligner (BWA). Variants are detected using the Broad Institute's Best Practice Variant Detection GATK toolkit.Median age was 68 years (range, 55–85), 50% were female, and MDS subtypes were as follows: RA/RCUD/RARS 13%, RCMD 16%, RAEB-1/2 20%, MDS/MPN & CMML-1/2 31%, and sAML 20%. Response was assessed using IWG 2006 criteria at 4 and 7 months after therapy initiation. Overall response was 48%; rate of CR (including marrow/cytogenetic CR) was 28%, any HI 20%, SD 22%, and no response 29%. The cohort was then dichotomized into “responders” and “non-responders,” with responders classified as those achieving CR or any HI. Baseline patient characteristics were similar between both groups, including average age at treatment initiation, disease subtypes, proportion of abnormal/complex karyotypes, and presence of common cytogenetic aberrations. Overall, the most frequently mutated genes include TET2/IDH1/IDH2, SRSF2, ASXL1, SF3B1, RUNX1, EZH2/EED/SUZ12, SETBP1, CBL, and PPIAF2. The highest rate of refractoriness was noted in mutants of TET2/IDH1/IDH2 (67%), SF3B1 (67%), U2AF1/2 (67%). We also identified several genes whose mutants were few but associated exclusively with refractory disease (100%), including KIT, ZRSR2, PRPF8, LUC7L2.We next applied a recursive partitioning algorithm to construct a decision tree for identifying the most pivotal mutations associated with response: we found mutant CBL and PPFIA2 to be strongly associated with response, whereas mutant U2AF1/2, SF3B1 and PRPF8 were strongly associated with refractoriness. Our final approach was to dichotomize the cohort by the presence/absence of each mutation/group of mutations, and response within mutant vs. wild type cases was compared. Among refractory cases, TET2/IDH1/IDH2 (26%) and SF3B1 (17%) were most frequently mutated; among responders, mutations in RUNX1 (19% vs. 4%]), CBL (14% vs. 0%), SRSF2 (23% vs. 9%), and SETBP1 (18% vs. 4%) were most frequent. When multiple genes were combined in “either-or” fashion, mutation in TET2, SF3B1, PRPF8, or LUCL71 was significantly associated with refractoriness (52%, p=.0287), whereas mutations of RUNX1, CBL, SRSF2, SETBP1, or PPFIA2 mutation was significantly associated with response (86%, p=.0001).Mutational patterns appear to predict response to standard hypomethylating agents. Identification of the most predictive genes could guide development of molecular maker-based selection of patients for hypomethylating agent therapy, but will require ongoing analysis and additional prospective testing for validation. Disclosures:Advani:Genzyme: Honoraria, Research Funding; Immunomedics: Research Funding. Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.

EZH2 Is Either Mutated or Downregulated in Patients with Loss of Heterozygosity of Chromosome 7/7q and Leads to Epigenetic Dysregulation Via Histone H3K27.

Abstract 228Chromosome 7 abnormalities occur either in isolation or in conjunction with other lesions (del(5q) or del/uniparental disomy (UPD)17p). To date the search for mutations affecting genes located on chromosome 7 has not been successful.By combination of metaphase and single nucleotide polymorphism array based karyotyping, in a cohort of 929 patients with myeloid malignancies (myelodysplastic syndromes (MDS): n=366; myelodysplastic/myeloproliferative neoplasms (MDS/MPN): n=108; MPN: n=206; secondary acute myeloid leukemia (sAML): N=167; primary (p)AML: n=82), loss of heterozygosity (LOH) in chromosome 7q was observed in 11.8% of MDS, 11.3% of MDS/MPN, 8.1% of MPN, 7.6% of sAML and 12.6% of pAML. In addition to somatic interstitial deletions (del7q) and a total loss of chromosome 7 (del7), somatic UPD(7q) was found: UPD(7q) was most frequently observed in MDS/MPN (4.9%). Similar to del(7/7q), UPD(7q) also conveyed a poor prognosis in terms of overall survival (OS). We hypothesized that cases with LOH7q may harbor a mutated gene which would explain clonal malignant evolution. For identification of such a hypothetical gene, we used two strategies: 1) application of a next generation sequencing platform and 2) targeted sequencing of candidate genes in intervals delineated by the presence of somatic microdeletions in specific patients. Both approaches demonstrated that the EZH2 gene can be affected by somatic mutations. Subsequently, screening led to detection of a total of 16 cases with EZH2 mutations, which were present in the majority of UPD(7q) patients, but in only 10% of cases with del(7q). In 4 cases (without LOH7q), we also found heterozygous EZH2 mutations. In addition to mutations, expression of EZH2 was found to be significantly decreased in patients with del(7/7q), likely due to haploinsufficiency, and surprisingly decreased in the entire cohort of patients, including 119/542 cases of AML.EZH2 mutations were observed in MDS/MPN (7.1%) and MPN (6.4%) but less frequently in AML. Further analyses of clinical phenotypes associated with the EZH2 mutation showed a significant negative impact on OS. In particular, the negative impact on OS was evident in the MDS/MPN group (p=0.0005) or in patients older than 60 years (p=0.0086). In the index case (sAML and UPD(7q)), 2 other mutations were detected: ASXL1 and TET2; all mutations were found at initial presentation prior to AML transformation. A concominant ASXL1 mutation was found in 3 other cases, while in 2 other patients, EZH2 mutations were found together with TET2 mutations.We have also investigated the pathogenic mechanisms resulting form EZH2 mutations. Leukemic cells from a patient with a homozygous EZH2 mutation easily initiated xenografts in NSG mice, which within 4 weeks of injection developed massive infiltration of spleen and liver with leukemic blasts. As expected, WB, ELISA and immunohistochemistry performed on these blasts revealed decreased H3K27 trimethylation compared to EZH2 wild type CD34 positive cells. A similar effect was observed in mutant cell line SKM-1 when compared to cell lines with wild type EZH2. When we investigated the effects of EZH2 on the gene expression profile in EZH2 mutant and WT cell lines, we found a significant increase in expression of HOXA9, known to be regulated by EZH2, along with PU.1, CDKN2A, IRF4, RASSF1 and CEBPA. We also investigated the effect of EZH2 mutations on the structural state of chromatin using a PCR-based method using non-denatured chromatin. We set up primers prior to and/or at the beginning of the first exons of MYC, CDKN2A and PU.1 to asses the chromatin plasticity. The EZH2 mutation was associated with more efficient amplification of these genes, suggestive of a relaxed, transcriptionally active state of DNA on the corresponding sites.In sum, mutations of EZH2 are present in patients with MDS/MPN and sAML. The mutations are mostly homozygous and are not commonly associated with del(7/7q). However, decreased expression of wild type EZH2 may be responsible for the pathogenesis associated with del(7/7q). Additionally, they convey a negative prognostic impact on OS. Mechanistically, decreased H3K27 trimethylation mediates downstream effects, such as persistent expression of various genes like HOXA9 and others. Mutations of EZH2 link the genomic instability to epigenetic dysregulation of gene expression. Disclosures:Maciejewski:Celgene: Speakers Bureau; Alexion: Speakers Bureau; Celgene: Research Funding; Eisai: Research Funding; NIH: Research Funding; AA & MDS International Foundation: Research Funding.

Distinction of Early and Late Molecular Events In Patients with Myelodysplastic Syndromes (MDS) Who Progressed to Acute Myeloid Leukemia (AML),

Abstract 3566MDS patients (pts) are destined to either progress to more aggressive disease or die of MDS-related complications. The International Prognostic Scoring System (IPSS) and other systems assess the survival and risk of evolution to acute myelogenous leukemia (AML), based in part on specific cytogenetic abnormalities. However, heterogeneity still exists even within cytogenetic groups, in part due to cryptic lesions, making predictions imperfect. In this respect, we and others have demonstrated that single nucleotide polymorphism array (SNP-A) karyotyping may improve the diagnostic yield, and that previously cryptic lesions can improve prognostic resolution of cytogenetic testing when compared to metaphase cytogenetics (MC) alone. Despite the recent identification of a variety of somatic mutations in MDS, their therapeutic value is unproven, and because of conflicting findings as to the risk for many mutations, they have not been routinely introduced into diagnostic algorithms.Mutational events may constitute suitable biomarkers that could strengthen current prognostic schemes. In addition, malignant progression is associated with acquisition of new molecular defects, which may impact our understanding of the molecular pathogenesis of MDS and allow for distinction of ancestral from facilitating defects. We hypothesized that serial cytogenetic testing for a comprehensive spectrum of mutations and cytogenetic defects would reveal which molecular event is associated with progression.We have analyzed a cohort of 76 MDS pts, 38 of whom experienced progression to AML (median age=68.5, IPSS = 0.5) matched to 38 pts (median age=69, IPSS =0.5) who did not experience progression. In addition to MC, we compared these groups for the presence of selected mutations, including TP53, RUNX1, TET2, ASXL1, DNMT3A, IDH1/2, NPM1, CBL and EZH2. The progressor group (PG) consisted of 20 (53%) low and intermediate-1 risk patients; the non-progressor group (NPG) had 22 (62%). In 60% of PG and 49% NPG at presentation, normal cytogenetics were present, while 16% and 14% had poor-risk cytogenetics, respectively. In addition, in 13% of PG and 11% of NPG, del7/7q was present. CBL, TP53, NPM1, ASXL1, IDH1, IDH2, RUNX1, DNMT3A, TET2 and EZH2 mutations were detected in (for PG vs. NPG): 0 (0/33) vs. 9% (3/35) p=1; 0 (0/29) vs. 5% (1/20) p=.4; 0 (0/33) vs N/A, 20% (6/30) vs. 18% (6/34) p=1; 9%(3/32) vs. 0 (0/39) p=.08; 0 (0/25) vs 0 (0/36) p=1; 0% (0/26) vs. 16% (1/6) p=.18; 7% (2/27) vs. 11% (4/37) p=1; 19% (4/21) vs. 37% (13/35) p=.23; and 5% (1/19) vs. 6% (2/32) p=1, respectively. We conclude that these pt groups had similar rates of mutations. We subsequently compared the cytogenetic and mutational status between initial presentation and at AML progression in the PG group. Progression was associated with occurrence of new cytogenetic lesions in 41% (14/34) while it remains stable in 53% (18/34) of pts. The most common new cytogenetics defects associated with evolution included trisomy 8, monosomy 7/del7q and monosomy 18. In 1, 2, and 1 pts, progression was associated with acquisition of CBL, TP53, and RUNX1 mutations, respectively. In contrast in 5, 3, 2 and 4 pts, ASXL1, IDH1, DNMT3A and TET2 mutations were present already at presentation. New mutations were detected upon leukemic evolution in 10/14 patients with abnormal MC and 3/14 with normal MC.We conclude that ASXL1 and TET2 (and perhaps IDH1 and DNMT3A) constitute early events while acquisition of TP53, CBL and possibly RUNX1 were events leading to progressive, advanced disease. Disclosures:Afable:Alexion: Speakers Bureau. Sekeres:Celgene Corporation: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Amgen: Membership on an entity’s Board of Directors or advisory committees. Maciejewski:Celgene: Speakers Bureau; Alexion: Speakers Bureau; Celgene: Research Funding; Eisai: Research Funding; NIH: Research Funding; AA & MDS International Foundation: Research Funding.

Aplastic Anemia & MDS International Foundation (AA&MDSIF): Bone Marrow Failure Disease Scientific Symposium 2010

Aplastic anemia (AA) and myelodysplastic syndromes (MDS) are a heterogeneous group of rare hematological disorders belonging to the Bone Marrow Failure (BMF) syndromes. The Aplastic Anemia and Myelodysplastic Syndromes International Foundation (AA&MDSIF) is a non-profit organization dedicated to supporting patients and families living with a BMF disease. They work to bring investigators together in a collaborative manner. This article summarizes key presentations from the last AA&MDSIF scientific symposium held in Bethesda, Maryland on March 2010.